Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.


Logo for

J. Biol. Chem. 281 (50): 38812-38824

© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Orexin-1 Receptor-Cannabinoid CB1 Receptor Heterodimerization Results in Both Ligand-dependent and -independent Coordinated Alterations of Receptor Localization and Function*

James Ellis, John D. Pediani, Meritxell Canals, Sandra Milasta, , and Graeme Milligan1

Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom

Abstract: Following inducible expression in HEK293 cells, the human orexin-1 receptor was targeted to the cell surface but became internalized following exposure to the peptide agonist orexin A. By contrast, constitutive expression of the human cannabinoid CB1 receptor resulted in a predominantly punctate, intracellular distribution pattern consistent with spontaneous, agonistindependent internalization. Expression of the orexin-1 receptor in the presence of the CB1 receptor resulted in both receptors displaying the spontaneous internalization phenotype. Single cell fluorescence resonance energy transfer imaging indicated the two receptors were present as heterodimers/oligomers in intracellular vesicles. Addition of the CB1 receptor antagonist SR-141716A to cells expressing only the CB1 receptor resulted in re-localization of the receptor to the cell surface. Although SR-141716A has no significant affinity for the orexin-1 receptor, in cells co-expressing the CB1 receptor, the orexin-1 receptor was also re-localized to the cell surface by treatment with SR-141716A. Treatment of cells co-expressing the orexin-1 and CB1 receptors with the orexin-1 receptor antagonist SB-674042 also resulted in re-localization of both receptors to the cell surface. Treatment with SR-141716A resulted in decreased potency of orexin A to activate the mitogen-activated protein kinases ERK1/2 only in cells co-expressing the two receptors. Treatment with SB-674042 also reduced the potency of a CB1 receptor agonist to phosphorylate ERK1/2 only when the two receptors were co-expressed. These studies introduce an entirely novel pharmacological paradigm, whereby ligands modulate the function of receptors for which they have no significant inherent affinity by acting as regulators of receptor heterodimers.

Received for publication March 16, 2006. Revision received September 29, 2006.

* This work was supported by the Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Davidson Bldg., University of Glasgow, Glasgow G12 8QQ, Scotland, UK. Tel.: 44-141-330-5557; Fax: 44-141-330-4620; E-mail: g.milligan{at}

Activation-Dependent Subcellular Distribution Patterns of CB1 Cannabinoid Receptors in the Rat Forebrain.
K. Thibault, D. Carrel, D. Bonnard, K. Gallatz, A. Simon, M. Biard, S. Pezet, M. Palkovits, and Z. Lenkei (2013)
Cereb Cortex 23, 2581-2591
   Abstract »    Full Text »    PDF »
Autocrine Endocannabinoid Signaling through CB1 Receptors Potentiates OX1 Orexin Receptor Signaling.
M. H. Jantti, J. Putula, P. M. Turunen, J. Nasman, S. Reijonen, C. Lindqvist, and J. P. Kukkonen (2013)
Mol. Pharmacol. 83, 621-632
   Abstract »    Full Text »    PDF »
Physiology of the orexinergic/hypocretinergic system: a revisit in 2012.
J. P. Kukkonen (2013)
Am J Physiol Cell Physiol 304, C2-C32
   Abstract »    Full Text »    PDF »
The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55.
J. Kargl, N. Balenga, G. P. Parzmair, A. J. Brown, A. Heinemann, and M. Waldhoer (2012)
J. Biol. Chem. 287, 44234-44248
   Abstract »    Full Text »    PDF »
OX1 Orexin/Hypocretin Receptor Signaling through Arachidonic Acid and Endocannabinoid Release.
P. M. Turunen, M. H. Jantti, and J. P. Kukkonen (2012)
Mol. Pharmacol. 82, 156-167
   Abstract »    Full Text »    PDF »
Novel Role for Proteinase-activated Receptor 2 (PAR2) in Membrane Trafficking of Proteinase-activated Receptor 4 (PAR4).
M. R. Cunningham, K. A. McIntosh, J. D. Pediani, J. Robben, A. E. Cooke, M. Nilsson, G. W. Gould, S. Mundell, G. Milligan, and R. Plevin (2012)
J. Biol. Chem. 287, 16656-16669
   Abstract »    Full Text »    PDF »
Functional Homomers and Heteromers of Dopamine D2L and D3 Receptors Co-exist at the Cell Surface.
C. Pou, C. Mannoury la Cour, L. A. Stoddart, M. J. Millan, and G. Milligan (2012)
J. Biol. Chem. 287, 8864-8878
   Abstract »    Full Text »    PDF »
Psychopharmacology of the endocannabinoids: far beyond anandamide.
F. Pamplona and R. Takahashi (2012)
J Psychopharmacol 26, 7-22
   Abstract »    Full Text »    PDF »
Differential Modulation of Brainstem Phosphatidylinositol 3-Kinase/Akt and Extracellular Signal-Regulated Kinase 1/2 Signaling Underlies WIN55,212-2 Centrally Mediated Pressor Response in Conscious Rats.
B. M. Ibrahim and A. A. Abdel-Rahman (2012)
J. Pharmacol. Exp. Ther. 340, 11-18
   Abstract »    Full Text »    PDF »
The role of the endocannabinoid system in the neuroendocrine regulation of energy balance.
F. J. Bermudez-Silva, P. Cardinal, and D. Cota (2012)
J Psychopharmacol 26, 114-124
   Abstract »    Full Text »    PDF »
Developing Chemical Genetic Approaches to Explore G Protein-Coupled Receptor Function: Validation of the Use of a Receptor Activated Solely by Synthetic Ligand (RASSL).
E. Alvarez-Curto, R. Prihandoko, C. S. Tautermann, J. M. Zwier, J. D. Pediani, M. J. Lohse, C. Hoffmann, A. B. Tobin, and G. Milligan (2011)
Mol. Pharmacol. 80, 1033-1046
   Abstract »    Full Text »    PDF »
Heteromultimerization of Cannabinoid CB1 Receptor and Orexin OX1 Receptor Generates a Unique Complex in Which Both Protomers Are Regulated by Orexin A.
R. J. Ward, J. D. Pediani, and G. Milligan (2011)
J. Biol. Chem. 286, 37414-37428
   Abstract »    Full Text »    PDF »
AT1R-CB1R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II.
R. Rozenfeld, A. Gupta, K. Gagnidze, M. P. Lim, I. Gomes, D. Lee-Ramos, N. Nieto, and L. A. Devi (2011)
EMBO J. 30, 2350-2363
   Abstract »    Full Text »    PDF »
Dopamine D1-histamine H3 Receptor Heteromers Provide a Selective Link to MAPK Signaling in GABAergic Neurons of the Direct Striatal Pathway.
E. Moreno, H. Hoffmann, M. Gonzalez-Sepulveda, G. Navarro, V. Casado, A. Cortes, J. Mallol, M. Vignes, P. J. McCormick, E. I. Canela, et al. (2011)
J. Biol. Chem. 286, 5846-5854
   Abstract »    Full Text »    PDF »
International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid Receptors and Their Ligands: Beyond CB1 and CB2.
R. G. Pertwee, A. C. Howlett, M. E. Abood, S. P. H. Alexander, V. Di Marzo, M. R. Elphick, P. J. Greasley, H. S. Hansen, G. Kunos, K. Mackie, et al. (2010)
Pharmacol. Rev. 62, 588-631
   Abstract »    Full Text »    PDF »
Allostery at G Protein-Coupled Receptor Homo- and Heteromers: Uncharted Pharmacological Landscapes.
N. J. Smith and G. Milligan (2010)
Pharmacol. Rev. 62, 701-725
   Abstract »    Full Text »    PDF »
Formation of {micro}-/{kappa}-opioid receptor heterodimer is sex-dependent and mediates female-specific opioid analgesia.
S. Chakrabarti, N.-J. Liu, and A. R. Gintzler (2010)
PNAS 107, 20115-20119
   Abstract »    Full Text »    PDF »
Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery.
T. Kenakin and L. J. Miller (2010)
Pharmacol. Rev. 62, 265-304
   Abstract »    Full Text »    PDF »
Signal transduction of the CB1 cannabinoid receptor.
G. Turu and L. Hunyady (2010)
J. Mol. Endocrinol. 44, 75-85
   Abstract »    Full Text »    PDF »
Ligand- and Heterodimer-Directed Signaling of the CB1 Cannabinoid Receptor.
B. D. Hudson, T. E. Hebert, and M. E. M. Kelly (2010)
Mol. Pharmacol. 77, 1-9
   Abstract »    Full Text »    PDF »
Hetero-oligomerization of CCR2, CCR5, and CXCR4 and the Protean Effects of "Selective" Antagonists.
D. Sohy, H. Yano, P. de Nadai, E. Urizar, A. Guillabert, J. A. Javitch, M. Parmentier, and J.-Y. Springael (2009)
J. Biol. Chem. 284, 31270-31279
   Abstract »    Full Text »    PDF »
Cannabinoids for clinicians: the rise and fall of the cannabinoid antagonists.
H. Butler and M. Korbonits (2009)
Eur. J. Endocrinol. 161, 655-662
   Abstract »    Full Text »    PDF »
Growth Hormone Secretagogues and Growth Hormone Releasing Peptides Act As Orthosteric Super-Agonists but Not Allosteric Regulators for Activation of the G Protein G{alpha}o1 by the Ghrelin Receptor.
K. A. Bennett, C. J. Langmead, A. Wise, and G. Milligan (2009)
Mol. Pharmacol. 76, 802-811
   Abstract »    Full Text »    PDF »
Biochemical and Electrophysiological Characterization of Almorexant, a Dual Orexin 1 Receptor (OX1)/Orexin 2 Receptor (OX2) Antagonist: Comparison with Selective OX1 and OX2 Antagonists.
P. Malherbe, E. Borroni, E. Pinard, J. G. Wettstein, and F. Knoflach (2009)
Mol. Pharmacol. 76, 618-631
   Abstract »    Full Text »    PDF »
The Action and Mode of Binding of Thiazolidinedione Ligands at Free Fatty Acid Receptor 1.
N. J. Smith, L. A. Stoddart, N. M. Devine, L. Jenkins, and G. Milligan (2009)
J. Biol. Chem. 284, 17527-17539
   Abstract »    Full Text »    PDF »
Bioluminescence Resonance Energy Transfer Studies Reveal Constitutive Dimerization of the Human Lutropin Receptor and a Lack of Correlation between Receptor Activation and the Propensity for Dimerization.
R. Guan, X. Feng, X. Wu, M. Zhang, X. Zhang, T. E. Hebert, and D. L. Segaloff (2009)
J. Biol. Chem. 284, 7483-7494
   Abstract »    Full Text »    PDF »
Endocannabinoid-Mediated Control of Synaptic Transmission.
M. Kano, T. Ohno-Shosaku, Y. Hashimotodani, M. Uchigashima, and M. Watanabe (2009)
Physiol Rev 89, 309-380
   Abstract »    Full Text »    PDF »
Conserved Polar Residues in Transmembrane Domains V, VI, and VII of Free Fatty Acid Receptor 2 and Free Fatty Acid Receptor 3 Are Required for the Binding and Function of Short Chain Fatty Acids.
L. A. Stoddart, N. J. Smith, L. Jenkins, A. J. Brown, and G. Milligan (2008)
J. Biol. Chem. 283, 32913-32924
   Abstract »    Full Text »    PDF »
Morphine Desensitization, Internalization, and Down-Regulation of the {micro} Opioid Receptor Is Facilitated by Serotonin 5-Hydroxytryptamine2A Receptor Coactivation.
J. F. Lopez-Gimenez, M. T. Vilaro, and G. Milligan (2008)
Mol. Pharmacol. 74, 1278-1291
   Abstract »    Full Text »    PDF »
Constitutive Activity of the Cannabinoid CB1 Receptor Regulates the Function of Co-expressed Mu Opioid Receptors.
M. Canals and G. Milligan (2008)
J. Biol. Chem. 283, 11424-11434
   Abstract »    Full Text »    PDF »
Allosteric Transinhibition by Specific Antagonists in CCR2/CXCR4 Heterodimers.
D. Sohy, M. Parmentier, and J.-Y. Springael (2007)
J. Biol. Chem. 282, 30062-30069
   Abstract »    Full Text »    PDF »
Protean Agonism at the Dopamine D2 Receptor: (S)-3-(3-Hydroxyphenyl)-N-propylpiperidine Is an Agonist for Activation of Go1 but an Antagonist/Inverse Agonist for Gi1,Gi2, and Gi3.
J. R. Lane, B. Powney, A. Wise, S. Rees, and G. Milligan (2007)
Mol. Pharmacol. 71, 1349-1359
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882